This invention relates to the preparation of tissues for cytometric analysis and observation. More particularly a liquid medium is described for preparing animal tissues for automated or microscopic analysis in which the cell nuclei are separated into discrete, non-agglomerated units. The medium and procedure is specifically tailored for flow cytometry.
In order for flow cytometry to be useful in studies of the proliferation kinetics and carcinogenesis of mammalian cells, a consistent, rapid method is necessary for the preparation of single cells or nuclei suspensions from solid tissues. Much work has been accomplished utilizing ascites tumors, cell cultures and hemopoetic tumors. Since these samples comprise single cells, preparation for cytometric analysis is simply a matter of staining the cells for DNA content while avoiding cell clumping. Sample preparation of solid tumors or tissues for flow cytometric analysis, however, becomes much more difficult, since the cells must be separated from each other completely in order to minimize false DNA histograms generated by two or more cells adhering to one another.
There is generally no acceptable procedure for the disaggregation of cells from tissues. The ideal technique, therefore, must be determined by trial and error in which the best method of disaggregation is only applicable for a specific tissue in only one species. This problem is amplified in human tumor biopsy analysis. An added problem of scar tissue build-up due to either surgical or radiation therapies can cause inconsistent results in multiple biopsies of the same patient.
Several preparative methods have been used for the production of single cells. The most popular procedures have utilized enzymes with the goal of recovering viable cells which are representative of the whole tissue. Some of the enzymes which have been used include trypsin for murine squamous-cell carcinoma, pepsin for metastatic human tumors, and combinations of trypsin-collagenase; see Noel, J. S. et al, "The Dissociation of Transplantable Tumors", J. Histochem Cytochem 25:544 (1977). Other methods have applied nonenzymatic, chemical procedures or physical techniques with the main goal to recover a maximum number of single cells for flow cytometric studies.
In general, these preparative methods are multistep processes which require a customized treatment on each type of tissue under analysis to insure maximum dissociation. Thus there is the disadvantage that each procedure is tissue specific. Also, the reproducibility of the majority of these methods has not been firmly established. Furthermore, cell dissociation is not always complete, and the intepretations of flow cytometric DNA histograms are suspect because of cell aggregation creating false DNA values. These cell aggregates also make microscopic pattern recognition difficult in that the edge boundaries of aggregated cells cannot always be clearly defined.
If one is interested in quantitating nuclear DNA in a single cell, then nuclear isolation would be sufficient. The standard biochemical procedures for nuclear isolation from tissues usually require homogenization and centrifugation steps which have been found to lead to incomplete tissue dissociation, release of DNA from destroyed nuclei and clumping of centrifuged nuclei. These procedures are unacceptable for flow cytometric measurements. Hypotonic solutions, always used with single cell preparations but not tissue, without and with the nonionic surfactant, nonidet P 40 (NP40) have been used to isolate nuclei from single cells for flow cytometric DNA analysis with propidium iodide. A hypertonic saline solution with NP40 has been utilized to obtain nuclei from solid tumors; see Vindelov, "Flow Microfluorometric Analysis of Nuclear DNA In Cells From Solid Tumors And Cell Suspension"; Virchows Arch [Cell Pathol] 24:227 (1977).
The objective of the present invention is to provide a dependable, rapid method for isolating fluorochrome stained nuclei from normal and cancerous tissues. This one-step procedure of combination nuclear isolation and fluorochrome DNA staining does not require a centrifugation step, therefore, nuclear clumping is avoided. A wide range of tissues may be utilized.
Accordingly it is an object of the present invention to provide materials and methods for measuring DNA in tissue cells by flow cytometry utilizing a one step combination nuclear isolation-DNA fluorochrome staining medium and procedure. The advantages of this procedure are that the disclosed nuclear isolation medium (NIM) provides a simple, one step procedure that takes only 5-10 min. to obtain a representative sample. This allows for the preparation of multiple samples from the same tissue in order to define more accurately the characteristics of the whole tissue. Moreover, with easily dissociated tissues, one can compare the single cell and tissue nuclear isolates to see if their DNA distributions are equivalent. In the majority of samples, there is minimal sample debris which results in consistent, high resolution DNA histograms in the 1-2% coefficient of variation range regardless of the tissue analyzed. Finally, since the NIM is easily prepared and the disaggregation procedure is a single step process, the whole preparation technique can be standardized, thus allowing accurate comparison of data between laboratories.
Besides the DNA quantitation there are a number of useful parameters in the cell nucleus to which the present invention is well adapted. First of all, nuclear volume may be a useful parameter in characterizing tumor development as shown for erythroleukemic cells. Furthermore, one of the most important criteria for the detection, classification, and staging of tumor cells is nuclear area. For example, the mean nuclear area for cells in carcinoma in situ is about three times that of normal squamous cell nuclei. Endometrial adenocarcinoma nuclei show an increase in area which is directly proportional to the grading scale of these tumors. Also, there is a significant increase in nuclear area in ectocervical when compared to endocervical tumors.
By utilizing high resolution DNA fluorescent measurement with electronic nuclear volume, two parameter scattergrams can be generated in order to develop a fingerprint technique for the detection and classification of tumors. This correlative analysis would be especially useful in solid tumor studies where single cell preparations are difficult to obtain. In general, there are a number of antigens, receptors and enzymes that may be expressed in the nuclei. Certain histocompatability antigens are expressed in the nuclear membrane. Probably the most important site in the cell to look for estrogen and progesterone receptors is in the nucleus. Finally, there are several important enzymes in the nucleus. One of these is found in NIM isolated liver nuclei where 5'-ATPase levels increase significantly in tumor cells. These and other applications of the procedures of the present invention will be apparent to those skilled in the art.